Much excitement has been generated in the technical, scientific and lay communities about the potential benefits arising from the developments of genetic engineering technology. Most frequently, public attention and praise are focused on the pharmacological and public health benefits associated with genetic engineering. Examples of the genetically engineered pharmaceutical products that have been given kudos include genetically engineered tissue plasminogen activator and, more recently, erythropoietin.
It is understandable that these pharmaceutical and public health related achievements have garnered much of the public attention surrounding genetic engineering. However, application of genetic engineering technology to nonpharmaceutical related areas holds the same excitement and promise as application of genetic engineering technology to the public health sector. In particular, the application of genetic engineering tools to agricultural products, both plant and animal, holds out the exciting prospect of disease and pest resistant animals and plants. In this connection, great energies have been spent in these fields as well as public health fields to perfect or optimize the genetic engineering methods used by scientists to create these beneficial fruits of genetically engineered products.
This invention is not the type of genetically engineered discovery that appears on the front pages of newspapers, on the radio or on television. The "sex appeal" that is required to achieve such notoriety is not a part of this invention. Rather, this invention is principally directed to the methodology of genetic engineering. It is envisioned that the application of the methodology taught by this invention will someday result in the creation of a genetically engineered plant, animal or drug that will achieve the type of notoriety which results in extensive media coverage.
Many lay individuals who otherwise understand and, indeed, marvel at the benefits genetic engineering holds for society, do not fully appreciate the high degree of technical skill required to establish a scientific methodology or protocol that permits the actual genetic manipulation of cells. At the most basic level, genetic engineering is the transfer or movement of the "chemical blueprint" of cells, as encoded in nucleic acids, from one cell in which such a chemical blueprint naturally occurs to another cell, called the host cell, that does not naturally contain the chemical blueprint. It is important that this transfer occur in such a way that the host cell can accurately read and express the chemical blueprint even though the blueprint is not its own.
One of the many technical difficulties of genetic engineering is to find a way to move the chemical blueprint, called deoxyribonucleic acid (DNA), from the cell in which it naturally occurs into the host cell. This problem of DNA movement required to transfect or transform a host cell is particularly acute in the case of plant host cell transformation, although the problem is certainly also prevalent in the transformation of other cellular life forms.
Various methodologies have been introduced into the genetic engineering field to facilitate this transfer of DNA from one cell to another. Included in such transfer technologies are the methods of electroporation, high velocity microprojectiles, polyethylene glycol (PEG) transformation, calcium phosphate, microinjection and Aqrobacterium-mediated transformation. Problems, particularly with DNA transformation of cells especially plant cells and most especially monocot plant cells such as corn (maize), are extant with all these methodologies.
For example, electroporation is many times an inefficient and highly costly methodology. Although electroporation is a reliable procedure, it results in pronounced loss of cell viability (3, 7) caused by, among other factors, destruction of the cell membrane and/or of cell wall integrity. Microprojectiles are cumbersome to use, costly and, as their name implies, damaging to cell membranes and/or cell walls causing significant loss of cell viability by virtue of the fact that a bullet-like projectile is being "shot" into the host cell. Aorobacterium-mediated DNA transformation is a painstaking process. In addition, it is not useful for monocot plant species generally because certain species are not susceptible to infection or transformation by Aqrobacterium bacteria, a step necessary for subsequent DNA transfer. The PEG methodology for host cell transfection is an extremely difficult methodology over which a genetic engineering researcher has somewhat minimal control and this methodology is highly toxic to cells generally. In addition to the difficulties associated with the aforementioned methodologies, all DNA transformation techniques must address the problem of introducing foreign DNA into a host cell so that the foreign DNA manifests some significant stability in the host cell thereby achieving the ultimate aim of genetic transformation--to stably express the transformed DNA chemical in a host cell. This is referred to as stable transformation of the host.
Achieving stable host transformation is a problem that extends across all forms of cellular life. However, the problem is especially acute in plant cells. Unlike animal cells that have no cell walls, plant cells have cellulosic cell walls. It is a characteristic of these cell walls that they are very difficult to penetrate. Accordingly, transformation of plant cells has proved more difficult than transformation of other cells because scientists attempting to transform nonplant cells need not deal with the complex problems of moving DNA across the cellulosic plant cell wall. Thus, it is easier to move DNA into an animal cell than a plant cell. Scientists have surmounted this problem by modifying plant cells so that the cellulosic cell wall is removed providing direct access to the plant cell membrane. (Because animal cells lack cell walls, membranes form the outer boundary of animal cells.) Plant cells lacking cell walls are termed protoplasts or naked cells. Because the cell wall is removed from these plant cells, the ability to transform such cells with nonnative DNA is increased. In most cases, the cell walls regenerate in 6-24 hours.
The achievement of stable transformation is a relative criterion that is principally a function of the time in which foreign DNA is inside a host cell and in which the host cell maintains the ability to express the transformed DNA. In plants and in animals such stable transformation is best achieved by methods which lead to the incorporation of the foreign DNA into chromosomes. However, stable integration especially in animal cells can be achieved with certain vectors that replicate freely in animal cells without integration into chromosomes. Until stable transformation is achieved, the transformation is said to be transient. Obviously, any host cell that has been stably transformed has passed through a stage at which it could only be said that the host cell was transiently transformed. However, it must be appreciated that transient transformation of a cell (and transient expression of the transformed DNA) is not a guarantor that stable transformation will occur. Among the reasons for this is the relative unpredictability of biological systems.
The transformation technique of this invention is a simple and elegant protocol that uses two common, relatively inexpensive "off-the-shelf" chemicals in a combined or coordinated manner such that a synergistic effect between the two chemicals results as manifest by extremely efficient DNA transformation in all cells, most notably in plant cells and especially notably in monocot plant cells. Indeed, the two chemicals that are combined by this invention, a polycation compound and a cationic liposome compound, have been used individually to effect DNA transformation. Although other researchers have combined DNA transformation methodologies in the past, in particular combining the techniques of PEG and electroporation (29), the reports of the alleged success of this latter combination have been dubiously received by the scientific community. Regardless of that, the combination of the present invention results in a very efficient transformation, significantly greater than the PEG and electroporation combination even assuming that such a combination is effective. Thus, this invention teaches a methodology termed 2PC that permits transient and stable transformation of host cells.
This invention employs two common chemicals used in an extremely simple methodology that does not require sophisticated machinery or great expense. Accordingly, this invention has great application for all genetic engineering laboratory undertakings, whether in highly sophisticated molecular biology research labs such as are extant in major universities and corporations throughout the world, or whether in simple laboratory settings such as in a high school biology laboratory.
It is therefore an object of the present invention to provide a method for transforming a host cell. More particularly, it is an object of this invention to provide a method for transformation of plant cells, especially monocot plant cells.
It is a further object of this invention to provide a method of cell transformation that is applicable to all cells and that employs relatively low technology means thus providing for the use of this technology by a broad spectrum of laboratories.
It is a still further object of this invention to provide for transformed cells that have been transformed by the synergistic action of a polycation compound and a cationic liposome to effect both transient and stable transformation of the host cell.